Several conditions of the eye result in migration and proliferation of fibroblasts. Such conditions include injury to the tissue of the eye, retinal detachment, filtration surgery to treat glaucoma, other eye surgeries, and other insults to the tissue of the eye. These conditions are susceptible to treatment or mitigation by agents that inhibit scarring and fibroblast migration and proliferation. Migration and proliferation of fibroblasts resulting from two of these conditions, retinal detachment and filtration surgery to treat glaucoma, is well characterized. In vitro and in vivo model systems have been developed for proliferative vitreoretinopathy that can result from retinal detachment. Scarring and bleb formation that can result from filtration surgery to treat glaucoma has also been studied using in vitro and animal models. Results of tests of therapeutic agents in these in vitro and in vivo models correlate with clinical results in humans. Given the wide involvement of scarring and fibroblast proliferation in degradation of vision, there exists a need for agents that ameliorate this degradation by mechanisms which include the inhibition of scarring and fibroblast proliferation.
Proliferative Vitreoretinopathy
Severe proliferative vitreoretinopathy (PVR) occurs in approximately 10% of patients who develop retinal detachment, and is the major cause of unsuccessful retinal reattachment surgery, leading to persistent retinal detachment and permanent loss of vision. Clinically, patients with early stages of proliferative vitreoretinopathy show increased haze and turbidity in the vitreous gel, indicating the presence of proliferating fibroblasts, retinal pigmented epithelial cells (RPE), and glial cells. This early stage of proliferative vitreoretinopathy is classified as Grade A. As these cells attach to the extracellular matrix (ECM) proteins on the retinal surface, the edge of the retinal tear becomes rolled as the cells begin to contract (Grade B). Later, as epiretinal membrane contraction progresses, fixed retinal folds develop (Grade C; folds in one quadrant=C1; folds in two quadrants=C2; folds in three quadrants=C3). These are typically most prominent in the inferior equatorial region due to gravity causing proliferating cells to settle and attach to the retinal ECM proteins in these locations.
As the clinical disease progresses, it is typically associated with total retinal detachment and fixed retinal folds due to the contraction of epiretinal membranes in all four quadrants (Grade D). This may be accompanied by antero-posterior foreshortening of the retina, caused by adhesion, proliferation and contraction of epiretinal membranes over the anterior retina and ciliary body, dragging the peripheral retina over the pars plana, ciliary body, and even onto the posterior surface of the lens.
If the disease process continues, the geometry of the retinal detachment progresses from an open to a closed "funnel" configuration; basically a scarred stalk of retina extending from the optic nerve anteriorly through the middle of the vitreous cavity to its anterior attachment behind the lens (see FIGS. 1 and 2). Untreated, retinal detachment results in permanent blindness. Even with the best of treatment, the visual prognosis is markedly diminished once significant proliferative vitreoretinopathy (grade C2 or greater) develops.
Conventional scleral buckling surgery can be used to repair most rhegmatogenous retinal detachments which are not associated with or have only minimal proliferative vitreoretinopathy. Retinal detachment associated with significant proliferative vitreoretinopathy (Grade C2 or greater) has a poor surgical outcome with conventional surgical techniques alone. Over the past fifteen years, techniques have been developed which allow some of these previously inoperable eyes to be salvaged. The current treatment for proliferative vitreoretinopathy includes conventional surgical techniques to support the peripheral retina and anterior vitreous base, followed by microsurgical removal of the vitreous gel and epiretinal membranes. The removal of these fine membranes from the surface of the retina is accomplished manually with 20 gauge (0.9 mm diameter) picks, forceps, scissors, and automated suction cutting instruments.
Despite many recent technological advances, such as improvement of microsurgical instrumentation, endolaser intraoperative retinal photocoagulation, and long acting intraocular tamponade agents, successful surgical retinal reattachment is currently achieved in severe proliferative vitreoretinopathy (Grade C3 or greater) in only about two-thirds of cases. Despite surgical reattachment, the visual outcome is poor, with only about 40% of patients achieving vision of 5/200 or better. It is generally accepted that further advances will occur only when it is possible to pharmacologically modify vitreoretinal scarring.
The cells most commonly reported to comprise proliferative vitreoretinopathy membranes include fibroblasts, retinal pigment epithelial cells, and glial cells. These types of cells have been reported to adhere to ECM proteins, synthesize ECM proteins, and express cell surface receptors for ECM proteins.
There are at least three components in the progression of proliferative vitreoretinopathy which lead to retinal detachment: cell migration, proliferation, and contraction. In order to monitor each of these components, three different assay systems may be used. The systems employ cultured dermal fibroblasts: (1) Boyden microchemotaxis chambers may be used to assess cell migration, (2) direct counts of cultures of fibroblasts may be carried out to examine cell proliferation, and (3) the collagen gel shrinkage assay may be employed to assess contraction. The collagen gel shrinkage assay has also been used to analyze the mechanisms by which the cells contract and induce retinal detachment.
These in vitro systems have been used to test a number of antimetabolites as well as agents that affect the cytoskeleton, such as cytochalasin B, colchicine, and taxol. These cytoskeleton affecting agents are thought to affect all three components noted above, since cell migration, division, and contraction are all dependent on the activity of the cytoskeleton.
The activity of dermal fibroblasts in assay systems containing daunomycin, cytochalasin B, colchicine, and taxol has been examined (see Verdoorn et al, Arch. Ophthalmol., 104, 1216-1219 (1986)). Colchicine was found to be most effective at inhibiting gel contraction followed by taxol and cytochalasin B. Daunomycin was not found to inhibit contraction. All four drugs inhibited cell proliferation, with daunomycin and taxol being the most effective. Migration was most inhibited by daunomycin, followed by colchicine and taxol. Cytochalasin B had a minimal effect on cell migration. Taxol has been tested in the gel contraction model and shown to inhibit cell migration, proliferation, and contraction.
Animal Models of Proliferative Vitreoretinopathy
Rabbit models of proliferative vitreoretinopathy have been developed and are widely accepted as reasonable representations of the human clinical disease process. In particular, the rabbit model, reported in Chandler et al., Graefe's Arch. Clin. Exp. Ophthalmol., 224, 86-91 (1986) and Thresher et al., Graefe's Arch. Clin. Exp. Ophthalmol., 221, 192-198 (1984), is widely used. This model has been used to test a variety of agents for their ability to inhibit proliferative vitreoretinopathy and vitreoretinal scarring.
Prior experimental therapies have tested the effectiveness of a variety of toxic antimetabolites such as 5-fluorouracil, methotrexate, adriamycin, vincristine, mitomycin, cisplatin, bleomycin, cytocine arabinoside, daunomycin, retinol, colchicine, and aclacinomycin A to control the proliferation of the cells. Taxol reduces traction retinal detachment induced by injected chorioretinal fibroblasts following 0.5 .mu.g intravitreal injection. However, none of these compounds has come into common clinical use in part due to concerns over retinal toxicity.
Intraocular corticosteroid therapy as a potential treatment for proliferative vitreoretinopathy has also been investigated in a rabbit model. The incidence of retinal detachment was reduced from 93% to 75% when eyes were injected with triamcinolone acetonide. The proliferation of injected fibroblasts was reduced in treated eyes due to the steroid mediated inhibition of mitosis. Subsequent experiments, however, showed that intraocular steroids were unable to inhibit the contraction of epiretinal membranes.
Heparin, a glycosaminoglycan, has been shown to inhibit fibroblast-mediated contraction of type I collagen gels. In addition, heparin inhibits the proliferation of human scleral fibroblasts and retinal pigmented epithelial cells and has been shown to prevent the development of intraocular fibrin membranes in the standardized proliferative vitreoretinopathy rabbit model. In the studies in the rabbit model, however, increased postoperative bleeding was observed.
A synthetic peptide, RGDS (Arg-Gly-Asp-Ser) (SEQ ID NO:16) originally derived from the extracellular matrix protein fibronectin (described below) is known to be important in mediating cell adhesion. Studies have shown that this peptide can inhibit the adhesion of retinal pigmented epithelial cells to fibronectin, type I collagen, type II collagen, and lens capsule basement membrane (see Avery et al., Arch. Ophthalmol., 104, 1220-1222 (1986)). There is no indication that this peptide will act as an inhibitor of collagen gel contraction or in vivo experimental proliferative vitreoretinopathy.
In view of the current state of treatments available for retinal reattachment in patients experiencing severe proliferative vitreoretinopathy, there is a continuing need for pharmacological therapies which will suppress vitreoretinal scarring. In addition to effectively suppressing proliferative vitreoretinopathy, such a pharmacological therapy should avoid cytotoxicity or other significant side effects.
Glaucoma
Glaucoma is a condition characterized by increased intraocular pressure in the eye which, if left untreated, leads to blindness. The increase in the intraocular pressure can be initially treated with medications but eventually may need surgery to lower the pressure in the eye to prevent blindness. The common surgery performed is filtration surgery. This surgery lowers the intraocular pressure by creating an alternate outflow channel that allows the egress of aqueous humor (the fluid within the eye) from the inside of the eye to the subconjunctival space (under the surface covering of the eye). Failure of glaucoma filtration surgery may result in increasing intraocular pressure. The most common cause of failure of glaucoma filtration surgery is scarring. The scarring is typically caused by the proliferation of cells (fibroblasts) and fibrosis in the subconjunctival space. Scarring can lead to an increase in intraocular pressure. To inhibit this process, several agents including anti-neoplastic agents (cancer chemotherapy) and corticosteroids have been studied experimentally, both in vivo and in vitro.
Many pharmacologic agents, including mitomycin-C and 5-fluorouracil, have been used to modulate the wound healing which follows filtration surgery. Filtration surgery is most likely to fail in eyes that have undergone previous cataract or glaucoma surgery. Youth, African-American ancestry, and active inflammation in the eye are also associated with increased risk of failure of the filtration surgery. Mitomycin-C and 5-fluorouracil are commonly used under these conditions to improve the success of the filtration surgery. 5-Fluorouracil has the drawback of frequent post-operative injections and is associated with complications such as wound leakage and corneal epithelial defects. Mitomycin-C is convenient in that it only needs a single application at the time of surgery but it may cause excessive filtration and results in persistent low eye pressure and its associated problems including decreased vision. There is accordingly a demand for other therapies which will inhibit scarring and increase the success rate of filtration surgery, while avoiding the complications seen with 5-fluorouracil and mitomycin-C.
Animal Models of Glaucoma Filtration Surgery
Rabbit models of glaucoma filtration surgery have been developed and are widely accepted as models of human glaucoma filtration surgery. In particular, the rabbit model described by Bergstrom et al., in Arch. Ophthalmology, 109(12), 1725-1730 (1991), is widely used. This model has been used to test a variety of agents for their ability to inhibit scarring after glaucoma filtration surgery.
The rabbit provides an excellent model to study filtration bleb failure because the rabbit exhibits a prolific fibroblastic response after surgery. Typically, the scarring and fibroblast proliferation results in failure of the surgery in untreated rabbits in only 9-14 days due to an inhibition or blocking of fluid drainage with a resulting increase in intraocular pressure. The fibroblast proliferation observed after filtration surgery is similar to the vigorous recruitment of fibroblasts that occurs in proliferative vitreoretinopathy and in other insults and conditions of the rabbit eye.
The rabbit model has been used to test a variety of therapeutic agents including antimetabolites like mitomycin, bleomycin, 5-fluorouracil (5-FU), inhibitors of connective tissue formation such as .beta.-aminopropionitrile and D-penicillamine, and other compounds such as tissue plasminogen activator and corticosteroid drugs. Most of these agents are effective but have undesirable side effects. Mitomycin and 5-FU have shown activity in the rabbit model and have allowed investigation of various routes of administration. For example, 5-FU has been administered in a collagen matrix implant or in a sponge. Concern over toxicity has limited the use of these compounds in humans, however.
In view of the current state of treatments available for failure of glaucoma filtration surgery, there is a continuing need for pharmacological therapies which will prevent scarring and fibroblast proliferation in the area of bleb formation. In addition, to effectively diminish scarring and failure in glaucoma filtration surgery, such a pharmacological therapy must avoid cytotoxicity or other significant side effects.